1. Field of the Invention
The present invention relates to a novel angiogenesis inhibitor, LK68 whose amino acid sequence is identical with the human apolipoprotein(a) kringle domains IV36, IV37 and V38, more specifically, to an amino acid sequence of the LK68, a cDNA sequence encoding the LK68, a recombinant expression vector comprising the cDNA, a recombinant microorganism transformed with the recombinant expression vector and a novel use of the LK68 as an anticancer agent and a method for treating the angiogenesis-mediated disease.
2. Description of the Prior Art
Angiogenesis is a biological process of generating new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. It has been reported that new vessel growth is tightly controlled by many angiogenic regulators (see: Folkman, J., Nature Med., 1: 27-31, 1995a), and the switch of the angiogenesis phenotype depends on the net balance between up-regulation of angiogenic stimulators and down-regulation of angiogenic suppressors.
An imbalance of the angiogenic process has been shown to contribute to pathological disorders such as diabetic retinopathy, rheumatoid arthritis and psoriasis (see: Folkman, J., Nature Med., 1: 27-31, 1995a). Especially, both primary and metastatic tumors need to recruit angiogenic vessels for their growth (see: Folkman, J., New Engl. J. Med., 285:1182-1186, 1971; Folkman, J., J. Biol. Chem., 267:10931-10934, 1992). If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. There are many reports suggesting that inhibiting tumor angiogenesis should provide a practical approach to long term control of the disease. Blocking positive regulators of angiogenesis or utilizing negative regulators to suppress angiogenesis results in a delay or regression of experimental tumors. If the angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. Moreover, in the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system effectively. Therefore, therapies directed at control of the angiogenic process could lead to the abrogation or mitigation of these diseases.
Therefore, what is needed is a novel angiogenesis inhibitor which can inhibit the unwanted growth of blood vessels, especially into tumors. An anticancer agent comprising the angiogenesis inhibitor should be able to overcome the activity of endogenous growth factors in premetastatic tumors and prevent the formation of the capillaries in the tumors thereby inhibiting the growth of the tumors. The anticancer agent should also be able to modulate the formation of capillaries in other angiogenic processes, such as wound healing and reproduction. Finally, the anticancer agent and method for inhibiting angiogenesis should preferably be non-toxic and produce few side-effects.
Until now, at least 10 endogenous angiogenic inhibitors have been identified in the art (see: O""Reilly, M. S. et al., Cell, 88: 277-285, 1997). One such molecule is angiostatin, which consists of the plasminogen kringle I through IV(see: O""Reilly, M. S. et al., Cell, 79:315-328, 1994). When applied systemically, angiostatin powerfully inhibits both primary tumor growth and metastasis without toxicity, and angiogenesis induced by bFGF as well (see: O""Reilly, M. S. et al., Nature Med., 2:689-692, 1996). These anti-tumor effects were accompanied by a marked reduction of microvessel density within the tumor mass, indicating that suppression of angiogenesis was associated with the inhibition of tumor growth.
Kringles are protein structural domains composed of approximately 80 amino acids and three intramolecular disulfide bonds. Kringle structures are found in many proteins such as prothrombin (see: Walz, D. A. et al., Proc. Natl. Acad. Sci., U.S.A., 74:1969-1973, 1977), plasminogen(see: Ponting, C. P., Blood Coagul. and Fibrinolysis, 3:605-614, 1992), urokinase(see: Pennica, D. et al., Nature, 301:579-582 1983), hepatocyte growth factor(see: Lukker, N. A. et al., Protein Eng., 7:895-903, 1994), and apolipoprotein(xe2x80x9capoxe2x80x9d)(a)(see: McLean, J. W. et al., Nature, 330:132-137, 1987). These domains appear to be independent folding units, but their functional role is not yet known. The previous reports represent that the kringle structure can act as inhibitors of endothelial cell migration and proliferation during angiogenesis. Specifically, prothrombin""s kringle 2 and plasminogen""s kringle 1-4, and 5 have been shown to be anti-angiogenic(see: Ji, W. R. et al., FASEB J., 15:1731-1738, 1998a; Ji, W. R. et al., Biochem. Biophys. Res. Commun., 247:414-419, 1998b; Cao, Y. et al., J. Biol. Chem., 271:29461-29467, 1996; Cao, Y. et al., J. Biol. Chem., 272:22924-22928, 1997; Barendsz-Janson, A. F., J. Vasc. Res., 35:109-114, 1998; Lee, T. H. et al., J. Biol. Chem., 273:28805-28812, 1998).
Apolipoprotein(a), one of the proteins having kringle structures, is a candidate for a novel angiogenesis inhibitor. Apo(a) is covalently attached to apoB-100, the main protein component of low density lipoprotein(LDL) to form lipoprotein(a)(see: Fless, G. M., J. Biol. Chem., 261: 8712-8718, 1986). Elevated plasma concentration of Lp(a) represents a major independent risk factor for artherosclerosis(see: Armstrong, V. W. et al., Artherosclerosis, 62:249-257, 1986; Assmann, G., Am. J. Cardiol., 77:1179-1184, 1996; Bostom, A. G. et al., JAMA, 276:544-548, 1996). Although several pathogenic activities have been reported, the physiological role of apo(a) has not yet been established(see: Lawn, R. M. et al., J. Biol. Chem., 271:31367-31371, 1996; Scanu, A. M. and Fless, G. M., J. Clin. Invest., 85:1709-1715, 1990; Utermann, G., Science, 246:0904-910, 1989).
Apo(a) contains two types of kringle domains and an inactive protease-like domains: the first 37 kringle domains are xcx9c75% identical to plasminogen kringle IV, and the last kringle domain is 90% identical to plasminogen kringle V. Interestingly, the kringle IV-like domain is present in 15-40 copies in different human alleles of the apo(a) gene. In this regard, it is feasible to develop an inhibitor of tumor angiogenesis and growth employing the Apo(a) kringle structures.
In accordance with the present invention, the inventors have cloned and expressed the human apo(a) kringles containing IV36, IV37 and V38 as a recombinant protein LK68, and discovered that: the LK68 protein and its single kringles, LK6, LK7 and LK8, have an ability to overcome the angiogenic activity of endogenous growth factors such as bFGF in vitro; and they may be used as active ingredients of anticancer agents.
The first object of the invention is, therefore, to provide a novel LK68 protein consisting of human apo(a) kringle domains IV36, IV37 and V38, and cDNA encoding the LK68 protein.
The second object of the invention is to provide a novel recombinant vector containing the cDNA encoding human apo(a) kringle domains IV36, IV37 and V38.
The third object of the invention is to provide an anticancer agent which comprises the LK68 protein or its single kringles, LK6, LK7 and LK8, as an active ingredient.
The fourth object of the invention is to provide a method for treating angiogenesis-mediated disease by employing the LK68 protein.